The present invention relates to a protein that inhibits activation of transcription factor NFxcexaB.
More detailed, the present invention relates to p65-binding protein (called ReIA-associate inhibitor or abbreviated as RAI hereinafter) that binds to p65, which is a subunit of transcription factor NFxcexaB, and that inhibits transcriptional activity of NFxcexaB, a process for producing it, a cDNA encoding it, a vector comprising the cDNA, a host cell transformed with the vector, an antibody against the inhibitor, and a pharmaceutical composition comprising the inhibitor or the antibody.
Further, the present invention relates to recombinant production of these proteins (especially, in vivo production), a nucleotide encoding them, a vector for expression and duplication and treatment and/or prevention of adult respiratory distress syndrome (ARDS), asthma, Allograft rejection, inflammatory diseases (inflammatory arthritis, angitis etc.), ischemic diseases, autoimmune diseases including chronic rheumatism, metastasis and invasion of cancer, vasoreconstriction and diseases induced by NFxcexaB besides above.
An acceleration of expressing many and various genes is observed in inflammation. Such genes include ones encoding interleukin, transcription factors, cohesive molecules, and factors in coagulation system etc. NFxcexaB which is a transcription factor has been said to relate to the transcription of such genes mostly.
It has been known that transcription factor NFxcexaB is expressed in cytoplasm. The transcription and induction of gene due to NFxcexaB-like proteins may be caused by activation of the protein. By such an activation, it becomes possible to translocate the transcription factor prepared in advance from cytoplasm to nucleus.
It is known that this translocation is controlled by phosphorylation and degradation of suppressor protein which is called for IxcexaB.
Transcription factor NFxcexaB was isolated from mature B cells in the form of binding to 10 nucleotide sequence motif in xcexa light chain enhancer for the first time. Therefore, NFxcexaB was thought to be specific for the generating stage of matured B cells. However, NFxcexaB-like proteins have been identified in a lot of cells, so it is shown that such a factor relates to induction of gene transcription generally. Such fact has been confirmed by functional identification of an active type NFxcexaB-binding position in some inducing genes.
NFxcexaB is a heterodimer consisting of a subunit of 50 kDa (p50) and a subunit of 65 kDa (p65).
NF kappa B (NFxcexaB), which is a nuclear factor, is a sequence-specific DNA-binding protein complex which regulates the expression of viral genomes, including the human immunodeficiency virus (HIV), and a variety of cellular genes, particularly those involved in immune and inflammatory responses. The members of the NFxcexaB family in mammalian cells include the proto-oncogene c-Rel, p50/p105 (NFxcexaB1), p65 (RelA), p52/p100 (NFxcexaB2), and RelB etc.
All of these proteins share a conserved 300 amino acid region of homology known as the Rel homology domain (RHD), which is responsible for DNA binding, dimerization, and nuclear translocation of NFxcexaB.
In most cells, Rel family members can form hetero- and homodimers with distinct specificities in various combinations. A common feature of the regulation of transcription factors belonging to Rel family is their sequestration in the cytoplasm as inactive complexes with a class of inhibitory molecules known as IKBs. Treatment of cells with a variety of inducers such as phorbol esters, interleukin 1, tumor necrosis factor-xcex1 (TNF-xcex1), viral infection and many mitogens and cytokines etc. results in the dissociation of the cytoplasmic complexes and translocation of free NFxcexaB into the nucleus. The dissociation of the cytoplasmic complexes is known to be triggered by the phosphorylation and subsequent degradation of the IKB proteins. Such a degradation exposes the nuclear localization sequence in the remaining NFxcexaB heterodimer, leading to nuclear translocation and subsequent binding of NFxcexaB to DNA regulatory elements within NFxcexaB target genes. The p65 subunit is frequently detected in NFxcexaB complexes and has a strong transcription activation potential. p65 dimerizes with other NFxcexaB family members and activates gene expression via its potent transactivation domain.
The object of the present invention is to provide a novel protein that inhibits transcriptional activity of NFxcexaB (NFxcexaB inhibitor), a process for producing it, a cDNA encoding it, a vector comprising the cDNA, a host cell transformed with the vector, an antibody against the inhibitor, and a pharmaceutical composition comprising the inhibitor or the antibody.
The present inventor et al. have thought that novel inhibitors may exist besides IxcexaB which was known as an NFxcexaB inhibitor. From the results of focusing their attention on and an extensive studies of NFxcexaB, especially p65 subunit, the present inventor et al. have achieved to find out a novel NFxcexaB inhibitor, confirmed amino acid sequence, a cDNA sequence encoding it, function of the inhibitor and tissue-distribution etc. and then competed the present invention.
RelA-associated inhibitor of the present invention is regarded as nucleus factor and inhibits NFxcexaB-dependent transcription activation by inhibiting DNA-binding activity of NFxcexaB. The gene encodes RelA-associated inhibitor is a gene having a high homology to the C-terminal region of 53BP2 containing four consecutive ankyrin repeats and an SH3 domain.
RAI appeared to locate in nucleus with the same location pattern of TNF-xcex1-induced p65. In addition, RAI inhibits activation of NFxcexaB, but does not show any influence on p53-dependent transcription activation in the cells which is transformed temporarily. Therefore, RAI is a novel p65-binding protein which relates to another mechanism of NFxcexaB-dependent transcription regulation. The present inventor has confirmed these interaction in vitro with bacterially expressed fusion proteins and in vivo using immunoprecipitation/ Western blot assay.
In spite of its similarity to 53BP2, RAI did not shown any interaction with p53 in a yeast two-hybrid assay. The cDNA encodes this protein has a high structual homology to 200 amino acid at the C-terminal region of 53BP2 containing four ankyrin repeats and an SH3 domain that are important for protein-protein interactions.
In human, mRNA of RAI was specifically expressed in heart, placenta and prostate while it was markedly reduced in liver, skeletal muscle and peripheral blood leukocyte.
Recent rapid developments in techniques for constructing cDNAs and sequencing techniques have made it possible to quickly sequence a large amount of cDNAs. By utilizing these techniques, a process, which comprises constructing cDNAs library from various cells or tussles, cloning cDNAs at random, identifying the nucleotide sequences thereof, expressing novel polypeptides encoded by them to analyze its physiological function, is now in progress. Method of yeast two-hybridization has been known as one of such techniques.
By this technique, it becomes possible to identify a large amount of homologous proteins at the same time and to obtain the aimed protein only easily by inserting reporter genes such as fluorescent marker etc.
The present inventor has carried out experiments of such a yeast two-hybrid screen using NFxcexaB protein subunit p65 as a probe to find a novel factor that binds to a novel p65 and that inhibits an activity of p65 and then found out the factor of the present invention.
The sequence of RelA-associated inhibitor of the present invention has been confirmed to be novel according to a homology search. Only a sequence of RelA-associated inhibitor of the present invention is a novel one among some clones obtained by the process for the sequence of the present invention described hereinafter.
The amino acid sequence of RelA-associated inhibitor of the present invention is as shown in SEQ ID NO. 1. This amino acid sequence contains four ankyrin repeats and an SH3 domain, so it is regarded as a member of Rel family.
The present invention relates to
(1) a RelA-associated inhibitor comprising amino acid sequence shown in SEQ ID NO. 1,
(2) a cDNA encoding the RelA-associated inhibitor described above (1),
(3) a cDNA comprising a nucleotide sequence shown in SEQ ID NO. 2, and
(4) a cDNA comprising a nucleotide sequence shown in SEQ ID NO. 3.